Novel 8-(furan-2-yl)-3-substituted thiazolo [5,4-e][1,2,4] triazolo[1,5-c] pyrimidine-2(3H)-thione derivatives as potential adenosine A2A receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2010.02.048

Novel thiazolotriazolopyrimidine derivatives (23-33) designed as potential adenosine A_2A receptor (A_2AR) antagonists were synthesized. Molecular docking studies revealed that all compounds (23-33) exhibited strong interaction with A<

Full text of DOI:10.1016/j.bmc.2010.02.048

Bioorganic & Medicinal Chemistry 18 (2010) 2491–2500
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 8-(furan-2-yl)-3-substituted thiazolo [5,4-e][1,2,4] triazolo[1,5-c]
pyrimidine-2(3H)-thione derivatives as potential adenosine A2A receptor
antagonists
Chandra Bhushan Mishra, Sandeep Kumar Barodia, Amresh Prakash, J. B. Senthil Kumar,
*
Pratibha Mehta Luthra
Medicinal Chemistry Division, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, North Campus, Mall Road, Delhi 110 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel thiazolotriazolopyrimidine derivatives (23–33) designed as potential adenosine A2A receptor
(A2AR) antagonists were synthesized. Molecular docking studies revealed that all compounds (23–33)
exhibited strong interaction with A2AR. The strong interaction of the compounds (23–33) with A2AR in
silico was confirmed by their high binding affinity with human A2AR stably expressed in HEK293 cells
using radioligand-binding assay. The compounds 24–26 demonstrated substantial binding affinity and
selectivity for A2AR as compared to SCH58261, a standard A2AR antagonist. Decrease in A2AR-coupled
release of endogenous cAMP in treated HEK293 cells demonstrated in vitro A2AR antagonist potential
of the compounds 24–26. Attenuation in haloperidol-induced motor impairments (catalepsy and akine-
sia) in Swiss albino male mice pre-treated with compounds 24–26 further supports their role in the alle-
viation of PD symptoms.
Received 2 December 2009
Revised 20 February 2010
Accepted 23 February 2010
Available online 1 March 2010
Keywords:
A2AR
Antagonist
cAMP assay
Haloperidol
Ó 2010 Elsevier Ltd. All rights reserved.
Radioligand-binding assay
Thiazolotriazolopyrimidines
1. Introduction
PD, A2AR antagonists have also been implemented to afford neuro-
protection in various animal models of PD.10,11
Adenosine alters vitally imperative processes in the CNS1 and is
natural ligand at the four well-conserved adenosine receptor sub-
types viz. A1, A2A, A2B and A3 belonging to G protein coupled recep-
tors.2 Adenosine A2A receptor (A2AR), 45 kDa protein3 is mainly
expressed in the nigrostriatum (basal ganglia) co-localized with
dopamine D2 receptors on striatopallidal output neurons.4 A2ARs
Numerous potent and selective A2AR antagonists (Fig. 1) of
structural variability have been synthesized.12–16 However, xan-
thine compounds such as KW6002 were unsuccessful in clinical
trials17 and non-xanthine compounds such as SCH58261 suffered
from lower selectivity, poor solubility and poor pharmacokinetic
profile.18 Recently, thiazole derivatives have revealed their thera-
peutic potential in various diseases.19,20 Thiazolo [4,5-g] dihydroin-
dazoles, thiazolo [5,4-b] pyrimidine-5 (4H)-ones and thiazole
[5,4-f] quinazolin-9-ones have exhibited PI3 kinase modulatory,
HIV-integrase inhibitory and GSK-3 inhibitory activities, respec-
tively. Thiazolopyrimidine exhibited adenosine A3 receptor antag-
onist activity21 and monocyclic thiazoles have been reported to
possess potential A2AR antagonist activity.22
In the present work, we have designed novel thiazolotriazolo-
pyrimidine derivatives as A2AR antagonists based on non-xanthine
tricyclic pharmacophore structural necessities such as tricyclic ring
structure and furan ring.15 The pharmacophore model Hypo-A2A
established that minimal structural requirement features for
A2AR antagonistic activity and selectivity were aromatic ring (R),
positively ionizable group (P), hydrophobicity (H), and hydrogen
bond acceptor (L).23 We replaced the pyrazole ring of SCH58261,
a selective A2AR antagonist by thiazole ring, and two categories
of N-substituted thiazolotriazolopyrimidine derivatives were
are abundantly expressed within striatum in a subset of
c-ami-
no-butyric acid (GABA-ergic) output neurons, which coexpress
higher levels of the dopamine D2 receptor and project to the globus
pallidus (GP).5 A2AR antagonist activates nigro-striatal pathway via
D2 receptor leading to antiparkinsonian effect.6 Parkinson disease
(PD) is neurodegenerative disorder caused by the degeneration of
dopaminergic neurons in nigrostriatal region of brain, and distin-
guished by malfunctioning of motor coordination apparent as tre-
mor and rigidity of the limbs and trunk.7 A2ARs have been
recognized as potential therapeutic target for the treatment of
PD.8 Blockade of the A2ARs in striatopallidal neurons diminished
postsynaptic effects of dopamine depletion, and in turn reduced
the motor deficits of PD.9 Besides providing symptomatic relief in
* Corresponding author. Tel.: +91 11 27666272x231; fax: +91 11 27666248.
E-mail addresses: pmluthra@acbr.du.ac.in, pmlsci@yahoo.com (P.M. Luthra).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.048

2492
C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
NH2
N
N
N
N
NH2
O
N
N
N
N
N
N
Cl
O
SCH58261
CGS 15943
NH2
N
N
N
N
N
N
N
N
S
S
O
N
N
N
O
Synthesized compound 25
SCH 63390
Figure 1. Structure of selective A2AR antagonist and synthesized compound.
prepared, one with flexible up to 4-carbon homologation, and
other substituted and unsubstituted planar aromatic ring to give
compounds (23–33). In silico interaction of the compounds (23–
33) with A2AR was carried using autodock analysis. In vitro affinity
and selectivity was determined by radioligand-binding assay in
membranes isolated from stably transfected HEK293 cells with hu-
man A2AR. Decrease in endogenous cAMP concentrations in treated
HEK293 cells and restoration of locomotor activity in haloperidol-
induced mice model was measured for most selective compounds
possessing strong binding affinity to appraise their potential as
A2AR antagonists.
monocyclic thiazole compounds 4-ethoxymethylene-imino-3-
substituted-2-thioxo-1,3-thiazole-5-carbonitriles (12–22), which
were used as key intermediates in cyclization reactions. The forma-
tion of Schiff’s bases (12–22) was confirmed on the basis of spec-
troscopic data. The disappearance of intense absorption band at
3230–3369 cmÀ1 for free amino group in IR spectra, and appear-
ance of singlet for N@CH in 1H spectra validated the formation of
Schiff’s bases (12–22). Condensation of monocyclic substrates
12–22 with 2-furoic acid hydrazide and isobutyric acid in toluene
for 4 h gave tricyclic thiazolo[4,5-b] pyrimidine compounds (23–
33) (Scheme 1). Disappearance of nitrile (–CN) absorption band
at 2205 cmÀ1 in IR spectra and appearance of singlet for N@CH of
pyrimidine ring in 1H NMR spectra appeared to confirm the forma-
tion of cyclized tricyclic compounds (23–33).
2. Results and discussion
2.1. Synthesis
2.2. Molecular docking study
Synthesis of thiazolotriazolopyrimidine compounds (23–33)
has been carried out according to Scheme 1. Concisely, the reaction
of substituted isothiocyanate, triethylamine, sulfur and malononit-
rile was carried at 0–5 °C for 1 h, then at room temperature for an
hour to give the carbonitrile compounds (1–11). The amino group
of the carbonitrile substrates (1–11) was extended with triethyl
orthoformate in toluene and p-toluenesulfonic acid (PTSA) to give
To explicate the interaction of thiazolotriazolopyrimidine
derivatives (23–33) with A2AR, molecular docking analysis of the
compounds 23–33 and known A2AR antagonist (SCH58261 and
ZM241385) were carried out with recently solved X-ray crystal
structure of human A2AR (PDB ID: 3EML). The A2AR (3EML) binding
cavity composed of TM2 (A63, I66), TM3 (A81, V84, L85), ECL2
R
NH2
CN
R
A
CN
CN
N
+ S +
NCS
1, 12, 23
2, 13, 24
3, 14, 25
4, 15, 26
5, 16, 27
6, 17, 28
7, 18, 29
8, 19, 30
9, 20, 31
10, 21, 32
11, 22, 33
C2H5
R
S
S
C3H7
C4H9
1-11
allyl
C6H5
B
p-FC6H5
p-ClC6H5
m-ClC6H5
o-IC6H5
p-IC6H5
m-IC6H5
R
N
N
O
C2H5
CH
CN
N
R
N
C
N
N
O
S
S
N
S
S
23-33
12-22
Scheme 1. Reagent and condition: (A) triethyl amine; (B) tritethylorthoformate, p-toluenesulfonic acid (PTSA), reflux; (C) furoic acid hydrazide, isobutyric acid, 50 °C.

C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
2493
(F168, E169), ECL3 (H 264, W268), TM5 (M174, M177) and TM6
(W246, L249, N253) domains. Docking simulation results showed
that thiazolotriazolopyrimidine derivatives (23–33) and SCH58261
shared a similar binding motif inside the transmembrane (TM)
region and extracellular loops of the human A2AR similar to the
co-crystallized ZM241385.24 The binding interaction of thiazolo-
triazolopyrimidine derivative 25 as one of the representatives
showed that the ring sulfur and exocyclic sulfur of thiazolo ring were
oriented towards the N253, M177 and V178 (Fig. 2). The involve-
ment of thiazolo sulfurs in H-bond interaction with amide moiety
of N253 was analogous to N-1 nitrogen at 7-position of ZM241385
and SCH58261 with A2AR. Residue N181 was involved in H-bond for-
mation with nitrogen of triazolo ring of thiazolotriazolopyrimidine,
SCH58261 and ZM241385. Further key receptor residues F168, E169
from ECL2 form aromatic stacking and hydrophobic polar interac-
tion with N1 position substituent of thiazolotriazolopyrimidine.
The receptor residues F183, H250 were involved in the formation
of H-bond with furan ring of thiazolotriazolopyrimidine, and the
hydrophobic interaction of residues V84, L85, V86, L249, F182 in
the vicinity of furan ring provided stability to hold the molecule in
the cavity.24,25 F168 (ECL2) provided aromatic stacking interaction
with the tricyclic core, H250, W246 and N253 were associated with
stabilizing the furan ring with mild polar interaction24,25 (Fig. 3).
Thiazolotriazolopyrimidine derivatives (23–33) possessed scaffolds
orientation and contacts almost similar to SCH58261 and
ZM241385. Moreover, the hydrophobic side chains of thiazolotriaz-
olopyrimidine derivative were the deciding factor for their sitting
conformation and affinity within the active site of A2AR. Thiazolo
ring N-substituent side chain ethyl, propyl, butyl, allyl and fluoro-
benzene showed higher binding affinity that could be related to
hydrophobicity and size of the substituent.
Figure 3. Molecular surface views of the active site of A2AR crystal structure,
compounds are represented as stick. Butyl (red), fluoro (deep salmon), propyl
(limon), SCH58261 (magenta), and ZM241385 (color by element i.e., CPK color) are
docked in the binding pocket. The molecular surface is color-coded by hydropho-
bicity properties. Basic amino acids (blue), acidic (red) and hydrophobic regions
(yellow) aliphatic (green) water molecule (red dot).
pyl derivative of thiazolotriazolopyrimidine (24) with greatly im-
proved binding affinity and selectivity with
A2AR (634-fold
selectivity over A1 receptor). Further extending the alkyl chain to
give butyl derivative of thiazolotriazolopyrimidine (25) showed
reasonably good affinity (0.12 nM) and tremendous selectivity
(1286-fold) compared to A1 receptor. However 3-carbon chain
2.3. Structure–activity relationship
with p-overlap in allyl derivative (26) led to excellent binding
affinity (0.016 nM) and selectivity (1250-fold over A1 receptor).
In other set of compounds, incorporation of N-1 aromatic ring
(phenyl) substituent (27) and p-fluorophenyl substitution (28) re-
duced binding affinity and selectivity. Chloro phenyl (m-, p-) and
iodo phenyl (o-, p-) substitution showed significant affinity with
A2AR and less selectivity over A1, though m-iodo phenyl (33) sub-
stitution displayed both high binding affinity and selectivity
(319.5-fold) towards A2AR. The results from in silico and in vitro
study accomplished that higher binding affinity of N-substituted
thiazolo ring with propyl, butyl, allyl and p-fluorobenzene with
A2AR could be due to their optimum topography at the binding site
as compared to ethyl (23), phenyl (27), m- and p-chlorophenyl (29,
30), and o-, m- and p-iodophenyl (31–33) side chains. High selec-
tivity of compounds 24–26 for A2AR could be related to specific
lipophlilic interaction in the active site. m-Substituted phenyl
derivative showed better affinity and selectivity for A2AR as com-
pared to phenyl and p-substituted phenyl derivatives. Overall, alkyl
derivatives (except ethyl) were more selective for A2AR than their
aromatic counterparts (27–33) probably due to topographical
parameter at the active site. Strong A1R affinity of ethyl-substi-
tuted thiazolotriazolopyrimidine indicates the conclusive role of
C-length for receptor binding (see Table 1).
The results of A2AR binding assay are expressed as inhibition
constants (Ki in nM). In the set of N-1 flexible side chains of thiaz-
olotriazolopyrimidine derivatives, ethyl substitution (23) exhibited
significantly higher binding affinity with A1 receptor (0.096 nM) as
compared to A2AR (3.56 nM). One carbon homologation gave pro-
In functional assay, binding of ligands to A2ARs promotes GPCR
mediated conformational change via Gas or Gai/o subfamily to
modulate the activity of adenylate cyclase and alter the concentra-
tion of cAMP. An antagonist generally interacts with A2AR via Gai/o
subfamily to inhibit the activity of adenylate cyclase leading to de-
crease in the cAMP concentration.26 In our study, most selective
compounds (24–26) treated with HEK293 cells stably transfected
with human A2RR reduced the endogenous cAMP concentrations
(0.10, 0.11 and 0.081 pmol/ml) further suggesting the A2AR antag-
onist activity of the compounds. The degree of reduction in endog-
enous cAMP concentration was significantly better than SCH58261
Figure 2. Hydrogen bond (H-bond) interactions (green dotted line) between
compound 25 A2AR binding site residues. Residues and compounds are shown in
stick representation. Nitrogen atoms are colored blue; oxygen atoms, red; and
sulfur atoms, yellow.

2494
C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
Table 1
Radioligand binding assay of thiazolotriazolopyrimidine compounds (23–33)
Compound
Structure
Ki (A2AR) nM
Ki (A1R) nM
A1/A2A
N
N
N
N
23
3.56
0.096
0.028
S
S
N
O
N
N
N
N
24
25
26
0.38
0.12
0.016
228.4
147.9
19.01
636.44
1286.09
1250.66
S
S
N
O
N
N
N
N
S
S
N
O
N
N
N
N
S
S
N
O
N
N
N
27
1.1
47.96
46.56
N
S
S
N
O
F
N
N
28
2.02
3.21
1.59
N
N
S
S
N
O
Cl
N
N
29
0.152
0.030
0.197
N
N
S
S
N
O
Cl
N
N
30
0.038
0.19
5
N
N
S
S
N
O

C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
2495
Table 1 (continued)
Compound
Structure
Ki (A2AR) nM
Ki (A1R) nM
A1/A2A
I
N
N
N
31
0.70
0.4
0.571
N
S
S
N
O
I
N
N
32
0.57
6.13
10.75
N
N
S
S
N
O
I
N
N
33
0.0082
2.62
319.5
N
N
S
S
N
O
NH2
N
N
N
SCH 58261
1.23
594.1
483
N
N
N
O
(0.25 pmol/ml), and higher than A2AR agonist NECA (0.75 pmol/ml)
and untreated cells (0.40 pmol/ml) (Table 2).
the extrapyrimidal symptoms of PD and is employed to study PD
in animal models. Catalepsy is condition characterized by muscular
rigidity of posture regardless of external stimuli and akinesia is the
inability to initiate movement. Blockade of A2ARs produces motor
stimulant effects and reverses catalepsy induced by dopamine
receptor blockade or by dopamine depletion.27 Previously, it has
been reported that A2AR antagonists improve motor impairment in
haloperidol-induced mice.28 The effect of A2AR antagonists on halo-
peridol-induced catalepsy has also been evaluated on primates.29 In
the present study, in vivo efficacy of the compounds 24–26 were as-
sessed at various doses (5, 10 and 20 mg/kg (Supplementary data) by
monitoring the restoration of locomotor activity (catalepsy and aki-
nesia) in haloperidol-induced mice for 120 min. Thiazolotriazolo-
pyrimidine derivatives (24–26) pre-treated haloperidol-induced
(2.5 mg/kg) mice exhibited attenuation of catalepsy and akinesia
(at doses 5, 10 and 20 mg/kg). The most effective dose for the
compounds 24–26 was 10 mg/kg at which catalepsy score was
comparable to SCH58261 (10 mg/kg (Table 3). The compound 24
2.4. Haloperidol-induced catalepsy and akinesia
Haloperidol, a dopamine D2 receptor antagonist blocks dopami-
nergic neurotransmission, induces catalepsy and akinesia causing
Table 2
cAMP functional assay of selected compounds (24–26)
Compounds
cAMP (pmol/ml)
Untreated cells
SCH58261
NECA
24
25
0.40
0.25
0.75
0.10
0.11
0.081
26
Table 3
Effect of selected compounds (24–26) on haloperidol-induced catalepsy
Compound
Dose (mg/kg bwt)
Time (min)
0
15
30
45
60
90
120
Saline
0
0
0
0
0
0
1% Acacia in saline
0
0
0
0
0
0
Haloperidol
SCH 58261
24
25
26
2.5
10
10
10
10
0
0
0
0
0
0.625 0.12
1.000 0.20
0
0.125 0.12
0.125 0.12
0.250 0.14
1.375 0.24
0.125 0.12
0.250 0.14
0.250 0.14
0.250 0.14
2.000 0.29
0.250 0.14
0.375 0.12
0.375 0.12
0.375 0.12
2.625 0.24
0.250 0.14
0.500 0.20
0.500 0.20
0.625 0.12
2.875 0.12
0.625 0.12
0.625 .12
0.750 0.14
0.750 0.14
0
0
0
0
Data represents the mean SEM; n = 4, p 60.05 compared with control.

2496
C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
pre-treated mice (10 mg/kg) exhibited catalepsy score of
0.625 0.12, which was similar to SCH58261 pre-treated mice.
However, the compounds 25 and 26 pre-treated mice (10 mg/kg)
displayed slightly higher catalepsy score (0.750 0.14) than
SCH58261 pre-treated mice. Vehicle groups saline and 1% acacia in
saline exhibited catalepsy score of 0 and haloperidol treated group
(2.5 mg/kg) represented catalepsy score of 2.875 0.12. The three
compounds showed comparative scores at different time intervals,
however, the compound 24 showed lowest catalepsy score after
120 min. Significant attenuation in catalepsy score was observed
in the compounds 24, 25 and 26 as compared to haloperidol treated
mice (Table 3).
fully characterized by TLC, IR, 1H NMR, 13C NMR, and Mass spec-
troscopy. IR spectra were performed on Spectrum BX FTIR, Perkin
Elmer. NMR spectra were recorded on AC Bruker 300 MHz spec-
trometer in CDCl3 or DMSO. Mass spectra were recorded on a
QSTAR XL LC–MS–MS, Applied Biosystem. Melting points were
determined using model KSPII, KRUSS, Germany. Elemental analy-
sis was performed on elementar analysensysteme. Column chro-
matography purifications were performed using silica gel Merck
100–200 mesh. Purity of the final step compounds was checked
by HPLC (Shimadzu model 8A) using acetonitrile (98%) + methanol
(2%) as mobile phase.
The compound 24 pre-treated (10 mg/kg) haloperidol-induced
mice showed akinesia score (39.50 5.4 s) comparable to
SCH58261 (10 mg/kg) pre-treated mice (32.25 1.4 s). Whereas,
compounds 25 and 26 (10 mg/kg) represented akinesia scores of
45.00 3.8 s and 49.25 5.8 s, respectively. The study demon-
strated that the latency period was maximum in 26, followed by
25 and 24, however, was significantly lower in these three com-
pounds as compared to haloperidol treated mice. Haloperidol trea-
ted mice (2.5 mg/kg) showed akinesia score of 119 1 s and
control groups saline and 1% acacia in saline exhibited 0 akinesia
score after 120 min. Evidently, our results demonstrated that com-
pound 24–26 antagonized the haloperidol-induced motor impair-
ments (catalepsy and akinesia) (Tables 3 and 4).
4.2. Synthesis of 4-amino-3-substituted-2-thioxo-1,3-thiazole-
5-carbonitrile (1–11)
Synthesis of compounds 1–11 have been carried according to
Luthra et al.29 Briefly, an equimolar mixture of isothiocyanate, mal-
ononitrile and sulfur powder in dimethylformamide was stirred in
ice bath. After 15 min, triethylamine was added drop-wise to the
mixture, and the reaction was continued to 4 h. The reaction mix-
ture was poured in water and precipitate was dissolved in absolute
ethanol to give pure crystalline 4-amino-3-substituted-2-thioxo-
2,3-dihydro-thiazole-5-carbonitrile derivatives (1–11).
4.2.1. 4-Amino-3-ethyl-2-thioxo-1,3-thiazole-5-carbonitrile (1)
Yield: 74%. White solid; mp: 205 °C. IR (KBr) 3308.95, 3227.11
(NH2), 2973 (alkyl), 2206.84 (CN), 1193.14 (C@S) cmÀ1 1H NMR
.
3. Conclusion
(CDCl3): d 1.27–1.32 (t, J = 7.2 Hz, 3H, –CH3), 4.22–4.29 (q,
J = 7.2 Hz, 2H, CH2), 6.38 (s, 2H, NH2). 13C NMR (CDCl3): 16.5, 45.6,
118.4, 158.7, 118.4, 158.7, 190.8. LC–MS: m/z 185 (M+), 186 (M+1).
We have reported the synthesis of new thiazolotriazolopyrimi-
dine derivatives (23–33) and demonstrated their potential as po-
tent and selective A2AR antagonists. In silico molecular docking
study showed that strong interaction of the compounds (23–33)
with A2AR were similar to standard antagonist SCH58261 as both
shared identical active site binding pocket. All the compounds dis-
played significantly high binding affinity with A2AR in in vitro radi-
oligand binding assay except three ( 23, 29 and 31). However,
compounds 24–26 displayed both higher binding affinity and
selectivity for A2AR in comparison to standard A2AR antagonist
SCH58261 and antagonized the A2AR-coupled release of endoge-
nous cAMP from HEK293 cells. Our results demonstrated that
blockade of A2ARs by thiazolotriazolopyrimidines (24–26) antago-
nize haloperidol-induced hypo-locomotor behavior (catalepsy
and akinesia) in vivo and produce motor stimulant effects to fur-
ther accentuate their potential for treatment of PD symptoms.
4.2.2. 4-Amino-3-propyl-2-thioxo-1,3-thiazole-5-carbonitrile
(2)
Yield: 85%. White solid; mp: 160 °C. IR (KBr), 3319.65, 3234.59
(NH2), 2205.95 (CN), 1198.14 (C@S) cmÀ1 1H NMR (CDCl3): d 1.03
.
(t, J = 7.5, 3H, CH3), 1.72–1.854 (m, 2H, CH2), 4.10 (t, J = 7.8 Hz, 2H,
CH2), d 4.83 (s, 2H, NH2). 13C NMR (CDCl3): 11.2, 20.3, 47.9, 118,
151.9, 186. LC–MS: m/z 199 (M+), 200 (M+1).
4.2.3. 4-Amino-3-butyl-2-thioxo-1,3-thiazole-5-carbonitrile (3)
Yield: 84%. Brown solid; mp: 155 °C. 3320.05, 3234.19 (NH2),
2208.95 (CN), 1199.14 (C@S) cmÀ1 1H NMR (300 MHz, CDCl3) d
.
0.97 (t, J = 7.2 3H, CH3), 1.353–1.476 (m, 2H, CH2), 1.66–1.74 (m,
2H, CH2), 4.16 (t, J = 7.8 Hz, 2H, CH2), 6.120 (s, 2H, NH2). LC–MS:
m/z 213 (M+), 214 (M+1).
4.2.4. 4-Amino-3-allyl-2-thioxo-1,3-thiazole-5-carbonitrile (4)
4. Experimental
4.1. General
Yield: 72%. Yellow solid; mp: 170–172 °C IR (KBr), 3313.65,
3224.89 (NH2), 2194.95 (CN), 1193.14 (C@S) cmÀ1 1H NMR
.
(CDCl3): d 4.88 (d, J = 4.8 Hz, 2H, CH2), 5.35 (d, J = 1.5 Hz, 2H,
CH2), 5.77–5.89 (m, 1H, CH), 6.50 (s, 2H, NH2). LC–MS: m/z 197
(M+), 198 (M+1).
TLC was carried out on commercially available TLC silica gel
(Silica Gel 60 F254) plates (Merck, Germany). The compounds were
Table 4
Effect of selected compounds (24–26) on haloperidol-induced akinesia
Compound
Dose (mg/kg bwt)
Time (min)
0
15
30
45
60
90
120
Saline
1% Acacia in saline
Haloperidol
SCH 58261
24
25
26
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2.5
10
10
10
10
78 12.6
111 5.4
0.75 0.75
6.00 3.5
4.75 2.7
6.00 3.5
112 5.2
118
2
119
1
119
1
0
0
0
0
6.50 2.3
7.75 3.3
15.00 3.6
21.25 4.3
16.50 4.4
16.50 3.8
24.25 4.4
33.00 2.8
26.25 3.4
26.25 4.9
33.50 2.9
41.50 3.1
32.25 1.4
39.50 5.4
45.00 3.8
49.25 .8
Data represents the mean SEM; n = 4, p 60.05 compared with control.

C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
2497
4.2.5. 4-Amino-3-phenyl-2-thioxo-1,3-thiazole-5-carbonitrile
(5)
Yield: 75%. Yellow solid; mp: 286 °C. IR (KBr): 3369, 3230 (NH2),
2204 (CN), 684 (C–S–C) cmÀ1. 1H NMR (300 MHz, CDCl3): d 5.90 (s,
2H, NH2) 7.12–7.47 (m, 5H, Ar). 13C NMR (75 MHz, CDCl3): d 112.2,
130.8, 131.1, 132.9, 133.7, 154.3, 188.1. LC–MS: m/z 233 (M+).
(t, J = 7.2 Hz, 3H, CH3), 1.46 (t, J = 7.2 Hz, 3H, CH3), 4.44 (q,
J = 7.2 Hz, 2H, CH2), 4.47 (q, J = 7.2 Hz, 2H, CH2), 8.332 (N@CH).
13C NMR (CDCl3): 12.4, 13.8, 41.6, 42.5, 65.2, 112.0, 161.5, 185.9.
LC–MS: m/z 242 (M+1).
4.3.2. 4-(Ethoxymethylene)-amino-3-(-propyl)-2-thioxo-1,3-
thiazole-5-carbonitrile (13)
4.2.6. 4-Amino-3-(p-fluorophenyl)-2-thioxo-1,3-thiazole-5-
carbonitrile (6)
Yield: 86%. Yellow viscous liquid; IR (KBr) 2952.36, 2854.68 (al-
kyl), 2208.00 (CN), 1265.12 (C@S) cmÀ1. 1H NMR (CDCl3): d 0.87 (t,
J = 7.2 Hz, 3H, CH3), 1.16 (t, J = 7.2 Hz, 3H, CH3), 2.27–2.38 (m, 2H,
CH2), 3.99 (q, J = 7.2 Hz, 2H, CH2), 4.22 (t, J = 7.5 Hz, 2H, CH2),
7.96 (s, 1H, N@CH). LC–MS: m/z 256 (M+1).
Yield: 65%. Yellow solid; mp: 219 °C. IR (KBr) 3352.61, 3248.85
(NH2), 2206.30 (CN), 1272.04 (C@S) cmÀ1 1H NMR (300 MHz,
.
CDCl3): d 5.968 (s, 2H, NH2), 7.29–7.37 (m, 4H, Ar). LC–MS: m/z
251 (M+), 252 (M+1).
4.3.3. 4-(Ethoxymethylene)-amino-3-(butyl)-2-thioxo-1,3-
thiazole-5-carbonitrile (14)
4.2.7. 4-Amino-3-(p-chlorophenyl)-2-thioxo-1,3-thiazole-5-
carbonitrile (7)
Yield: 85%. Yellow viscous liquid; IR (KBr) 2956.36, 2865.32 (Al-
kyl), 2212.26 (CN), 1268.66 (C@S) cmÀ1. 1H NMR (CDCl3): d 0.98 (t,
J = 6.6 Hz, 3H, CH3), 1.27–1.29 (m, 2H, CH2), 1.29 (t, J = 7.2 Hz, 3H,
CH3), 1.51–1.53 (m, 2H, CH2), 4.09 (q, J = 7.2 Hz, 2H, CH2), 4.46 (t,
J = 6.9 Hz, 2H, CH2), 8.13 (s, 1H, N@CH). LC–MS: m/z 270 (M+1).
Yield: 55%. Yellow solid; mp: 172–173 °C. IR (KBr) 3298.61,
3234.85 (NH2), 2204.30 (CN), 1244.04 (C@S), 744 (C–Cl) cmÀ1 1H
.
NMR (300 MHz, CDCl3) d 6.61 (s, 1H, NH2) 7.27 (d, J = 8.1 Hz, 2H,
Ar), 7.33 (d, J = 7.8 Hz. 2H, Ar). LC–MS: m/z 267 (M+), 268 (M+1).
4.2.8. 4-Amino-3-(m-chlorophenyl)-2-thioxo-1,3-thiazole-5-
carbonitrile (8)
4.3.4. 4-(Ethoxymethylene)-amino-3-(allyl)-2-thioxo-1,3-
thiazole-5-carbonitrile (15)
Yield: 56%. Brown solid; mp: 178–180 °C. IR (KBr) 3299.81,
Yield: 65%. Yellow crystals; mp: 122–124 °C. 2958.06, 2851.08
3236.85 (NH2), 2206 (CN), 1248.04 (C@S), 748 (C–Cl) cmÀ1 1H
.
(alkyl), 2206.56 (CN), 1268 (C@S) cmÀ1 1H NMR (CDCl3): d 1.44
.
NMR (300 MHz, CDCl3): d 5.92 (s, 2H, NH2), 7.26–7.51 (m, 4H).
(t, J = 7.2 Hz, 3H, CH3), 4.46 (q, J = 7.2 Hz, 2H, CH2), 4.79 (d,
J = 4.5 Hz, 2H, CH2), 5.25 (d, J = 0.9 Hz, 2H, CH2), 5.76–5.89 (m,
1H, CH), 8.26 (s, 1H, N@CH). LC–MS: m/z 254 (M+1).
LC–MS: m/z 267 (M+), 268 (M+1).
4.2.9. 4-Amino-3-(o-iodophenyl)-2-thioxo-1,3-thiazole-5-
carbonitrile (9)
4.3.5. 4-(Ethoxymethylene)-amino-3-(phenyl)-2-thioxo-1,3-
thiazole-5-carbonitrile (16)
Yield: 48%. Yellow solid; mp: 238 °C. IR (KBr) 3329.61, 3234.85
(NH2), 2201.30 (CN), 1276.04 (C@S) cmÀ1 1H NMR (300 MHz,
.
Yield: 55%. Yellow crystal; mp: 116 °C. IR (KBr) 2208.00 (CN),
CDCl3): d 4.58 (s, 2H, NH2), 7.37–7.70 (m, 4H, Ar). LC–MS: m/z
360 (M+1).
1250.18 (C@S) cmÀ1 1H NMR (CDCl3) d: 1.12 (t, J = 7.2 Hz, 3H,
.
CH3), 4.04 (q, J = 7.2 Hz, 2H, CH2), 7.17–7.26 (m, 2H, Ar), 7.46–
7.56 (m, 3H, Ar), 8.16 (s, 1H, N@CH). LC–MS: m/z 289 (M+), 290
(M+1).
4.2.10. 4-Amino-3-(p-iodophenyl)-2-thioxo-1,3-thiazole-5-
carbonitrile (10)
Yield: 45%. Red solid; mp: 228–230 °C. IR (KBr) 3328.61,
4.3.6. 4.3.64-(Ethoxymethylene)-amino-3-(p-fluorophenyl)-2-
thioxo-1,3-thiazole-5-carbonitrile (17)
3236.85 (NH2), 2201.30 (CN), 1278.04 (C@S) cmÀ1 1H NMR
.
(300 MHz, CDCl3): d 5.96 (s, 1H, NH2), 7.35 (d, 2H, Ar), 7.57 (d,
2H, Ar). LC–MS m/z 360 (M+1).
Yield: 55%. Yellow viscous liquid; IR (KBr) 2950.12, 2842.88 (al-
kyl), 2204.08 (CN), 1262.00 (C@S) cmÀ1. 1H NMR (CDCl3): d 1.27 (t,
J = 7.2 Hz, 3H, CH3), 4.18 (q, J = 7.2 Hz, 2H, CH2), 7.43 (d, J = 8.1, 2H,
Ar), 7.75 (d, 2H, J = 8.1, 2H, Ar), 8.03 (s, 1H, N@CH). 13C NMR
(CDCl3). LC–MS: m/z 308 (M+1).
4.2.11. 4-Amino-3-(m-iodophenyl)-2-thioxo-1,3-thiazole-5-
carbonitrile (11)
Yield: 45%. Red solid; mp: 230–232 °C. IR (KBr) 3328.62,
3236.85 (NH2), 2201.30 (CN), 1278.04 (C@S) cmÀ1 1H NMR
.
4.3.7. 4-(Ethoxymethylene)-amino-3-(p-chlorophenyl)-2-
thioxo-1,3-thiazole-5-carbonitrile (18)
(300 MHz, DMSO): d 7.61–7.26 (m, 3H, Ar), 7.95 (d, 1H, J = 8.7Hz,
Ar). LC–MS m/z 360 (M+1).
Yield: 52%, Yellow viscous liquid. IR (KBr), 2956.23, 2862.12 (al-
kyl), 2202 (CN), 1262 (C@S) cmÀ1 1H NMR (CDCl3): d 1.29 (t,
.
4.3. 4-(Ethoxymethylene) amino-3-substituted-2-thioxo-1, 3-
thiazole-5-carbonitrile (12–22)
J = 7.2 Hz, 3H, CH3), 4.19 (q, J = 7.2 Hz, 2H, CH2), 7.31–7.52 (m,
4H, Ar), 8.04 (s, 1H, N@CH). LC–MS m/z 323 (M+), 324 (M+1).
Compounds 12–22 have been synthesized according to Luthra
et al.30 Concisely, the carbonitrile compounds (1–11) were refluxed
with triethylorthoformate (equal equivalents) and PTSA (catalytic
amount) in toluene for 6 h. The reaction mixture was poured in
water, toluene layer was separated and washed with water, dried
with Na2SO4. The crude product was purified by column chroma-
tography to give viscous liquid compounds (11, 13, 14 and 17–
22) and solid products (12, 15 and 16). Solid compounds were
re-crystallized from absolute ethanol.
4.3.8. 4-(Ethoxymethylene)-amino-3-(m-chlorophenyl)-2-
thioxo-1,3-thiazole-5-carbonitrile (19)
Yield: 52%. Yellow viscous liquid, IR (KBr) 2942.23, 2844.43, (al-
kyl) 2202 (CN), 1265 (C@S). 1H NMR (CDCl3): d 1.16 (t, J = 6.9 Hz,
3H, CH3), 4.41 (q, J = 6.9 Hz, 2H, CH2), 7.47–7.53 (m, 3H, Ar), 7.80
(d, J = 8.1, 1H, Ar), 8.44 (s, 1H, N@CH). LC–MS: m/z 324 (M+1).
4.3.9. 4-(Ethoxymethylene)-amino-3-(o-iodophenyl)-2-thioxo-
1,3-thiazole-5-carbonitrile (20)
Yield: 54%. Yellow viscous liquid IR (KBr), 2952, 2802 (alkyl),
4.3.1. 4-(Ethoxymethylene)-amino-3-(ethyl)-2-thioxo-1,3-
thiazole-5-carbonitrile (12)
2205 (CN), 1257 (C@S) cmÀ1
. d 1.28 (t,
1H NMR (CDCl3):
J = 7.2 Hz, 3H, CH3), 4.20 (q, J = 7.2 Hz, 2H, CH2), 7.38–7.62 (m,
4H, Ar), 8.03 (s, 1H, N@CH). LC–MS (EI, 70 eV). m/z 415 (M+1).
Yield: 75%. Yellow crystal; mp: 116 °C. IR (KBr) 2962.13, 2852
(alkyl), 2209.28 (CN), 1228 (C@S) cmÀ1 1H NMR (CDCl3): d 1.27
.

2498
C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
4.3.10. 4-(Ethoxymethylene)-amino-3-(p-iodophenyl)-2-
thioxo-1,3-thiazole-5-carbonitrile (21)
J = 4.5 Hz, 1H, CH2). 5.29 (d, J = 0.9 Hz, 2H, CH2), 5.93–6.06 (m,
1H, CH), 6.66 (q, J = 1.5 Hz, 1H, Ar), 7.31 (d, J = 3.3 Hz, 1H, furan),
7.67 (s, 1H, Ar), 9.23 (1H, N@CH). 13C NMR (CDCl3): 48.3, 106.2,
112.2, 114.0, 119.9, 129.2, 138.7, 144.7, 145.4, 149.1, 159.5,
187.8. LC–MS: m/z 315 (M+), 316 (M+1). HPLC purity 100% Anal.
Calcd for C13H9N5OS2: C, 49.51; H, 2.88; N, 22.21; S, 20.33. Found:
C, 49.48; H, 2.81; N, 21.94; S, 20.28.
Yield: 54%. Yellow viscous liquid IR (KBr), 2952, 2802 (alkyl),
. d 1.29 (t,
2205 (CN), 1257 (C@S) cmÀ1 1H NMR (CDCl3):
J = 7.2 Hz, 3H, CH3), 4.28 (q, J = 7.2 Hz, 2H, CH2), 7.35 (d,
J = 8.4 Hz, 2H, Ar), 7.60 (d, J = 8.4 Hz, 2H, Ar), 8.12 (s, 1H, N@CH).
LC–MS: m/z 415 (M+1).
4.3.11. 4.3.11.4-(Ethoxymethylene)-amino-3-(m-iodophenyl)-
2-thioxo-1,3-thiazole-5-carbonitrile (22)
4.4.5. 8-(2-Thioxo-7(3-phenyl)-2-(2-furyl) thiazolo [4,3-e]1,2,4-
triazolo[1,5-c] pyrimidine (27)
Yield: 65%. Yellow viscous liquid IR (KBr) 2962.20, 2857 (alkyl),
Yield: 62%. White solid; mp: 298–299 °C. IR (KBr), 3059 (Aro-
2206 (CN), 1272 (C@S) cmÀ1
.
1H NMR (CDCl3): d 1.06 (t, J = 6.9 Hz,
matic CH), 1222 (C@S) cmÀ1 1H NMR (300 MHz, CDCl3): d 6.65
.
3H, CH3), 4.07 (q, J = 6.9 Hz, 2H, CH2), 7.49–7.51 (m, 3H, Ar), 7.79
(d, J = 8.1, 1H, Ar), 8.4 7 (s, 1H, N@CH) LC–MS: m/z 415 (M+1).
(d, J = 1.5 Hz, 1H,), 7.32 (d, J = 3.6 Hz, 1H furan), 7.42–7.68 (m,
6H, Ar, H), 9.13 (s, 1H, N@CH). LC–MS: m/z 351 (M+), 352 (M+1).
HPLC purity 100%.
4.4. Synthesis of 8-(2-thioxo-7-(3-substituted-2-(2-furyl)
thiazolo [4,e]-1,2,4-triazolo [1,5-c] pyrimidine (23–33)
4.4.6. 8-(2-Thioxo-7(3-p fluorophenyl)-2-(2-furyl) thiazolo [4,3-
e] 1,2,4-triazolo[1,5-c] pyrimidine (28)
Synthesis of compounds 23–33 have been carried according to
Shaker et al.31 with minor modifications. An equivalent mixture
of imino-ether derivatives (12–22) in toluene, 2-furoic acid hydra-
zide and isobutyric acid was stirred at 60 °C for 1–2 h. The reaction
continued with azeotropic removal of water. The precipitate was
filtered and washed with absolute hot ethanol and dried. The crude
product was purified by column chromatography and re-crystal-
lized to give pure tricyclic compound (23–33).
Yield: 75%. White solid; mp: 260 °C IR (KBr) 3036 (Ar, CH), 1260
(C@S) 1412 (C–F) cmÀ1 1H NMR (300 MHz, CDCl3): d 6.63 (q,
.
J = 1.8 Hz, 1H, Ar), 7.30–7.44 (m, 5H, Ar), 7.68 (t, J = 1.5 Hz, 1H,
Ar) 9.13 (S, 1H, N@CH). 13C NMR (CDCl3): 106.4, 112.2, 114.0,
116.8, 117.1, 130.4, 130.5, 138.9, 144.5, 145.4, 147.4, 149.1,
159.6, 188.4. LC–MS: m/z 369 (M+), 370 (M+1). HPLC purity 99.5%.
4.4.7. 8-(2-Thioxo-7(3-p-chlorophenyl.)-2-(2-furyl) thiazolo
[4,3-e] 1, 2 4-triazolo [1,5-c] pyrimidine (29)
4.4.1. 8-(2-Thioxo-7(3-ethyl)-2-(2-furyl) thiazolo [4,3-e] 1,2,4-
triazolo[1,5-c] pyrimidine (23)
Yield: 54%. White solid; mp: 280 °C IR (KBr) 1119 (C–Cl),
3033.21 (Ar, CH), 1262 (C@S) cmÀ1 1H NMR (300 MHz, CDCl3): d
.
Yield: 72%. White solid; mp: 248 °C IR (KBr) 2981.66, 2788.39
6.57 (s, 1H, furan), 7.10 (d, J = 7.8 Hz, 2H, Ar), 7.36 (d, J = 3.3 Hz,
1H, Ar), 7.61 (s, 1H), 7.91 (d, J = 7.8 Hz, 2H, Ar), 9.05 (s, 1H, N@CH).
13C NMR (CDCl3): 106.9, 112.3, 114.1, 116.8, 117.2, 130.6, 131.8,
139.0, 144.8, 145.9, 147.3, 149.2 161.2, 190.2. LC–MS: m/z 385
(M+), 386 (M+1). HPLC purity 100%.
(alkyl), 1253 (C@S), 3064.43 (Ar, CH) cmÀ1 1H NMR (300 MHz,
.
CDCl3): d 1.44 (t, J = 7.2 Hz, 3H, CH3). 4.62 (q, J = 7.2 Hz, 2H, CH2),
6.63 (q, J = 1.5 Hz, 1H, Ar), 7.31 (d, J = 3 Hz, 1H, Ar), 7.68 (s,
1H,Ar), 9.26 (s, 1H, N@CH). 13C NMR (CDCl3): 12.3, 41.9, 112.2,
113.9, 138.7, 144.7, 145.7, 147.1, 149.2, 159.1, 187.1. LC–MS: m/z
303 (M+), 304 (M+1). HPLC purity 100%.
4.4.8. 8-(2-Thioxo-7(3-m-chlorophenyl)-2-(2-furyl) thiazolo
[4,3-e] 1,2,4-triazolo[1,5-c] pyrimidine (30)
4.4.2. 8-(2-Thioxo-7(3-propyl)-2-(2-furyl) thiazolo [4,3-e] 1,2,4-
triazolo[1,5-c] pyrimidine (24)
Yield: 55%. White solid; mp 275 °C, IR (KBr) 1122 (C–Cl), 3035
(Ar, CH), 1265 (C@S) cmÀ1 1H NMR (300 MHz, CDCl3): d 6.64 (q,
.
Yield: 76%. White solid mp: 245 °C IR (KBr) 2965.13, 2330.96,
J = 1.8 Hz, 1H, Ar), 7.44 (d, J = 3 Hz, 1H Ar), 7.51 (d, J = 0.9 Hz, 1H,
Ar), 7.5 6–7.65 (m, 3H, Ar), 7.69 (s, 1H, Ar,) 9.20 (s, 1H, N@CH).
13C NMR (CDCl3): 106.8, 112.3, 114.1, 128.4, 130.6, 131.8, 138.9,
144.6, 145.3, 147.3, 159.3, 189.2. LC–MS: 385 (M+), m/z 386
(M+1). HPLC purity 100%.
2870.84 (alkyl), 1226 (C@S), 3071 (Ar, CH) cmÀ1 1H NMR
.
(300 MHz, CDCl3): d 1.04 (t, J = 7.5 Hz, 3H, CH3), 1.84–1.97 (m, 2H,
CH2), 4.50 (q, J = 7.5 Hz, 2H, CH2), 6.62 (q, J = 1.8 Hz, 1H, Ar), 7.30
(d, J = 2.7 Hz, 1H, Ar), 7.67 (d, J = 0.6 Hz, 1H, Ar), 9.22 (s, 1H, N@CH).
13C NMR (CDCl3): 11.2, 20.5, 48.2, 106.4, 112.2, 113.3, 138.5, 144.7,
145.4, 147.3, 149.4, 159.5, 187.9.LC–MS: m/z 317 (M+), 318 (M+1).
HPLC purity 100% Anal. Calcd for C13H11N5OS2: C, 49.19; H, 3.49;
N, 22.07; S, 20.21. Found: C, 48.97; H, 3.41; N, 21.87; S, 20.05.
4.4.9. 8-(2-Thioxo-7(3-o-iodophenyl)-2-(2-furyl) thiazolo [4,3-
e] 1,2,4-triazolo[1,5-c]pyrimidine (31)
Yield: 65%. White solid; mp: 316–317 °C IR (KBr), 3031 (Ar, CH),
1208 (C@S) cmÀ1 1H NMR (300 MHz, DMSO): d 6.75 (s, 1H, Ar),
.
4.4.3. 8-(2-Thioxo-7(3-butyl)-2-(2-furyl),thiazolo,[4,3-e]1,2,4-
triazolo[1,5-c] pyrimidine (25)
7.36–7.65 (m, 5H, Ar), 7.98 (s, 1H, Ar), 9.68 (s, 1H, N@CH). LC–
MS: m/z 477 (M+), 478 (M+1). HPLC purity 97.1%.
Yield: 84%. White solid; mp: 220 °C IR (KBr), 2961, 2927, 2856
(Butyl). 1190 (C@S) cmÀ1
.
1H NMR (300 MHz, CDCl3): d 1.00 (t,
4.4.10. 8-(2-Thioxo-7(3-p-iodophenyl)-2-(2-furyl) thiazolo [4,3-
e] 1,2,4-triazolo[1,5-c] pyrimidine (32)
J = 7.5, 3H, CH3), 1.40–1.53 (m, 2H, CH2), 1.79–1.89 (m, 2H, CH2),
4.50 (t, J = 7.5 Hz, 2H, CH2), 6.63 (q, J = 1.8 Hz, 1H, Ar), 7.29 (d,
J = 3.3 Hz, 1H, Ar), 7.67 (d, J = 0.6 Hz, 1H, Ar), 9.28 (s, 1H, N@CH).
13C NMR (CDCl3): 13.7, 20.0, 29.6, 46.6, 106.4, 112.2, 113.9,
138.5, 144.7, 145.4, 147.3, 149.4, 159.5, 187.8. LC–MS (m+1) LC–
MS: m/z 331 (M+), 332 (M+1). HPLC purity 100% Anal. Calcd for
C14H13N5OS2: C, 50.74; H, 3.95; N, 21.13; S, 19.35. Found: C,
50.54; H, 3.82; N, 21.02; S, 19.29.
Yield: 63%. White solid; mp: 302 °C IR (KBr), 3036 (Ar, CH), 1094
(C-I), 1259 (C@S) cmÀ1 1H NMR (300 MHz, CDCl3): d 6.63 (d,
.
J = 1.5 Hz, 1H, Ar), 7.32 (d, J = 3 Hz, 1H, furan), 7.38 (d, J = 8.7 Hz,
2H, Ar), 7.62 (d, J = 8.4 Hz, 2H, Ar), 7.68 (s, 1H, furan), 9.13 (s, 1H,
N@CH). 13C NMR (CDCl3): 106.6, 112.3, 114.1, 129.8, 130.3, 133.6,
136.4, 138.8, 144.6, 145.5, 147.29, 149.7, 159.8, 188.8. LC–MS: m/
z 477 (M+), 478 (M+1). HPLC purity 100%
4.4.4. 8-(2-Thioxo-7(3-allyl)-2-(2-furyl) thiazolo [4,3-e] 1,2,4-
triazolo [1, 5-c] pyrimidine (26)
4.4.11. 8-(2-Thioxo-7(3-m-iodophenyl)-2-(2-furyl) thiazolo
[4,3-e] 1,2,4-triazolo[1,5-c]pyrimidine (33)
Yield: 63%. White solid; mp: 253 °C IR (KBr), 1635 (C@C), 1262
Yield: 63%. White solid; mp: 312–313 °C IR (KBr), 3031.30 (Ar,
(C@S), 3033.21 (Ar, CH) cmÀ1
.
1H (300 MHz, CDCl3): d 5.16 (d,
CH), 1208 (C@S) cmÀ1 1H NMR (300 MHz, CDCl3): d 6.64 (d,
.

C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
2499
J = 1.5 Hz, 1H, Ar), 7.32–7.44 (m, 3H, Ar), 7.68 (s, 1H), 7.77 (s, 1H,
Ar), 7.92 (d, 1H, Ar), 9.17 (s, 1H, Ar). 13C NMR (CDCl3): 106.6,
112.3, 114.1, 127.9, 131.2, 136.2, 137.3, 138.9, 139.3, 144.7,
145.5, 149.6, 159.7,188.8.LC–MS (EI-70 eV): m/z 477 (M+), 478
(M+1). HPLC purity 99.7%.
respectively. Assays were performed in duplicates and compounds
were tested thrice. Data were fitted in one site competition-bind-
ing model for IC50 determination using the program GraphPad
Prism 4.0 (GraphPad Software, Inc., San Diego, CA) and Ki values
were calculated using Cheng and Prusoff formula.35
4.5. Molecular docking
4.7. Functional assay
Docking simulations were performed with AutoDock3.0.5.32
In the functional assay, cAMP concentrations were determined
using direct cAMP EIA kit (Assay designs, USA) in treated (com-
pounds 24–26) HEK293 cells (stably transfected with cDNA encod-
ing human A2AR). Cells (1 Â 106) were treated with 100 nM of
compounds (24–26), 100 nM SCH58261 and 100 nM of NECA for
24 h, 0.1 N HCl was added to release endogenous cAMP, centri-
fuged at 1000g for 5 min at 4 °C. cAMP concentrations were mea-
sured,36 and the results were compared with untreated cells
(representing basal level of cAMP).
The recently solved X-ray crystal structure of the human A2A
R
(pdb code: 3EML; 2.6-Å resolution) in complex with ZM241385
was retrieved from the RCSB Protein Data Bank and all heteroat-
oms were removed. The A2AR was set up for docking with standard
protocol. Polar hydrogens were added using PROTONATE utility
distributed with AutoDock3.0.5 then protein coordinates were
subjected to minimize with CHARMm force field potential.33,34
Steps (1000) of steepest descent followed by conjugate gradient
minimization until the RMS gradient of the potential energy was
less than 0.05 kJ molÀ1 ÅÀ1 performed the minimizations. Kollman
united atom charges and solvation parameters were added to the
final protein file. The known A2AR antagonists and thiazolotriazol-
opyrimidine derivatives were drawn on BUILDER module (Insight
II). DISCOVER molecular dynamics (CHARMm force field) was run
to carry the optimization of the ligands, which were saved in
mol2 format with the aid of BABEL programs (Pat Walters and Matt
Stahl, NCI). Full hydrogens were added to the ligands and Gastei-
ger-Marsili partial atomic charges were computed using the BABEL
program and saved in the PDBQ format. All possible flexible tor-
sions of the resultant ligand molecules were defined by using
AUTOTORS. The prepared ligands in PDBQ format were used as in-
put files for AutoDock3.0.5 in the next step. Docking simulation
was carried out using the Lamarckian Genetic Algorithm.32 For
binding energy, three terms were taken into account in the docking
step: the van der Waals interaction represented as a Lennard-Jones
12–6 dispersion/repulsion term, the hydrogen bonding repre-
sented as a directional 12–10 term, and the Coulombic electro-
static potential. The resulting docking orientations lying within
2.0 Å in the root-mean square deviation (rmsd) tolerance of each
other were clustered together represented by the result with the
most favorable free energy of binding. Finally, the obtained top-
posed docking conformations were subjected to post-docking en-
ergy minimization on Insight-II/Accelrys.33
4.8. Animals
Adult Swiss Albino male mice (4–6 weeks, 20–30 g) were pro-
cured from National Institute of Communicable Diseases, Delhi, In-
dia and were kept under controlled conditions of temperature
(22 1 °C), humidity (60 5%), and illumination (12 h light; 12 h
darkness) at the animal house, Dr. B.R. Ambedkar Centre for Bio-
medical Research, University of Delhi, Delhi, India. The experimen-
tal protocol met the National Guidelines on the ‘Proper Care and
Use of Animals in Laboratory Research’ (Indian National Science
Academy, New Delhi) and was approved by the Animal Ethics
Committee of the department. The procedures adhered to the
NIH Guidelines for the Care and Use of Laboratory Animals.
4.9. Drugs
Haloperidol and SCH58261 were purchased from Sigma Chem-
icals Co. (St. Louis, Mo) and Tocris, respectively, India. Double dis-
tilled filtered and deionized water (Milli-Q-system Waters,
Milford, MA) was used through out the study. Haloperidol,
SCH58261 and compounds 24–26 were dissolved in 1% acacia in
saline. Mice were divided into seven groups of four mice each.
SCH58261 (10 mg/kg) and compounds 24–26 (5, 10 and 20 mg/
kg) were administered intraperitoneally (ip) to each mice of the as-
signed group. Saline and 1% acacia in saline were injected to two
control groups. After 30minutes of pre-treatment, haloperidol
(2.5 mg/kg) was injected (ip) to one group and all pre-treated
groups.
4.6. Radioligand binding assay
Adenosine A2AR and A1R binding assays: [3H]ZM241385 and
[3H]DPCPX binding assays for adenosine A2AR and A1Rs, respec-
tively, were performed in stably transfected HEK293 cells (pro-
cured from National Center for Cell Sciences, Pune, India) with
4.10. Catalepsy
human A2AR and A1R. Briefly, 10
lated from were incubated with different concentrations (1 pM–
M) of compounds and 1 nM [3H]ZM241385 in 200
l incubation
buffer containing 50 mM Tris-Cl, 1 mM EDTA, pH 7.4 and 2.5 U/ml
adenosine deaminase. Adenosine A1R assays were performed on
l
g HEK293 cell membranes iso-
The inability of an animal to correct an externally imposed pos-
ture (Catalepsy score) was measured at different time intervals
with both limbs on a square wooden block (3 cm high) by placing
the animals on a flat horizontal surface.37 The length of time that
animals held the bar without any voluntary movement was re-
corded, with a cutoff time of 3 min. Catalepsy score of each mice
in a group was taken to compute the mean value of the group.38
1
l
l
10
l
g of HEK293 cell membranes expressing human adenosine
A1Rs and 1 nM [3H]DPCPX in 200
ll incubation buffer. Reactions
were carried out for 60 min at 26 °C and were terminated by rapid
filtration over 96-well plates equipped with GF/B filters (Milipore,
4.11. Akinesia
USA). Filters were washed three times with 300 ll of cold washing
buffer containing 50 mM Tris-Cl, 10 mM MgCl2, pH 7.4, air dried,
and radioactivity retained on filters were counted in 1450 LSC&
Luminescence counter (Wallac Microbeta Trilux, Perkin–Elmer,
USA). The Kd values for radioligands [3H]ZM241385 and [3H]DPCPX
were 0.99 and 1.79 nM, respectively, obtained by saturation bind-
ing assays. Non-specific binding for adenosine A2AR and A1R were
Akinesia was measured by noting the latency in seconds (s) of
the animals to move all four limbs and the test was terminated if
latency exceeded 180 s. Each animal was initially acclimatized
for 5 min on a wooden elevated (30 cm) platform (40 Â 40 cm)
used for measuring akinesia in mice. Using a stopwatch, the time
taken (s) by the animal to move all the four limbs was recorded.38
This exercise was repeated five times for each animal.
determined in the presence of 50 lM NECA and 50 lM CPA,

2500
C. B. Mishra et al. / Bioorg. Med. Chem. 18 (2010) 2491–2500
11. Shiozaki, S.; Ichikawa, S.; Nakamura, J.; Kitamura, S.; Yamada, K.; Kuwana, Y.
4.12. Statistical analysis
Psychopharmacol. 1999, 147, 90.
12. Caulkett, P. W. R.; Jones, G.; McPartlin, M.; Renshaw, N. D.; Stewart, S. K.;
Wright, B. J. Chem. Soc., Perkin Trans.1. 1995, 7, 801.
13. Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Pineda de Villatoro, M. J.; Zocchi, C.;
Dionisotti, S.; Ongini, E. J. Med. Chem. 1996, 39, 1164.
14. Zocchi, C.; Ongini, E.; Conti, A.; Monopoli, A.; Negretti, A.; Baraldi, P. G.;
Dionisotti, S. J. Pharmacol. Exp. Ther. 1996, 276, 398.
15. Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.; Monopoli, A.; Ongini, E.;
Varani, K.; Borea, P. A. J. Med. Chem. 2002, 45, 115.
The data for behavioural studies were statistically evaluated for
significance employing nonparametric analysis of variance (Krus-
kal-Wallis test) for catalepsy and akinesia employing statistical
package, Graph Pad Prism 4.0. Values of p 60.05 were considered
significant. Results are given as mean SEM values.
16. http://www.kyowa-kirin.co.jp/english/news/2009/e20090115_01.html.
17. Kisselgof, E.; Tulshian, D. B.; Arik, L.; Zhang, H.; Fawzi, A. Bioorg. Med. Chem.
Lett. 2005, 15, 2119.
Acknowledgements
18. Hirota, T.; Sasaki, K.; Tashima, Y.; Nakayaama, T. J. Heterocycl. Chem. 1991, 28,
263.
19. Nagamatsu, T.; Kinoshita, K.; Sasaki, K.; Nakayama, T.; Hirota, T. J. Heterocycl.
Chem. 1991, 28, 513.
20. Hirota, T.; Leno, K.; Sasaki, K. J. Heterocycl. Chem. 1986, 23, 1685.
21. Jacobson, M. A. Drug Dev. Res. 1996, 37, 131.
22. Andrew, G. C.; Tara, M. S.; Laura, L. R.; Axel, M.; Lawrence, W. D.; Wenguang, Z.;
Ian, H. Bioorg. Med. Chem. Lett. 2009, 19, 378.
23. Wei, J.; Wang, S.; Gao, S.; Dai, X.; Gao, Q. J. Chem. Inf. Model. 2007, 47, 613.
24. Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y. T.; Lane, J.
R.; Ijzerman, A. P.; Stevens, R. C. Science 2008, 322, 1211.
25. Catia, L.; Ippolito, A.; Michela, B.; Diego, D. B.; Dhuldeo, D. K.; Rosaria, V.; Karl,
N. K.; Gloria, C. Bioorg. Med. Chem. 2009, 17, 2812.
Chandra Bhushan Mishra, Amresh Prakash, J. B. Senthil Kumar,
Sandeep Kumar Barodia are thankful to Department of Science
and Technology, Delhi, Department of Biotechnology, Delhi, and
University Grant Commission, Delhi, India, respectively, for the
financial support. Authors are thankful to Council of Scientific
and Industrial Research, Delhi, India, for providing the funds to car-
ry this work.
Supplementary data
26. William, T.; John, F.; David, U. Curr. Opin. Biotechnol. 2005, 16, 655.
27. Jiang, F. C.; Rosario, M.; Francesco, I.; David, K. G.; Beatriz, C.; Marcelo, R.; Mark,
A. B.; Elizabeth, H.; Stephen Fink, J.; Malcolm, J. L.; Ennio, O.; Michael, A. S. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 1970.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.048.
28. Luthra, P. M.; Prakash, A.; Barodia, S. K.; Kumari, R.; Mishra, C. B.; Kumar, J. B.
Neurosci. Lett. 2009, 463, 215.
29. Varty, G. B.; Hodgson, R. A.; Pond, A. J.; Grzelak, M. E.; Parker, E. M.; Hunter, J. C.
Psychopharmacology 2008, 200, 393.
30. Luthra, P. M.; Mishra, C. B.; Jha, P. K.; Barodia, S. K. Bioorg. Med. Chem. Lett. 2010,
20, 1214.
31. Shaker, R. M. ARKIVOC 2006, XIV, 68.
32. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, K. R.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
33. Accelrys Inc., ForceField-Based Simulations. San Diego: Accelrys, Inc., 2002.
URL http://www.accelrys.com/doc/life/insight2000.1/ffbs/FF_SimulTOC.html.
34. Discover 3 User Guide, San Diego: MSI, USA, 1999.
35. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
36. Tijssen, P. Practice and Theory of Enzyme Immunoassays; Elsevier: Amsterdam,
1985.
37. Costall, B.; Naylor, R. J. Eur. J. Pharmacol. 1974, 27, 46.
38. Luthra, P. M.; Barodia, S. K.; Raghubir, R. J. Neurosci. Methods 2009, 178, 284.
References and notes
1. Ribeiro, J. A.; Sebastiao, A. M.; De Mendonca, M. Prog. Neurobiol. 2002, 68, 377.
2. Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K. N.; Linden, J.
Pharmacol. Rev. 2001, 53, 527.
3. Barrington, W. W.; Jacobson, K. A.; Hutchison, A. J.; Williams, M.; Stiles, G. L.
Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6572.
4. Svenningsson, P.; LeMoine, C.; Fisone, G.; Fredholm, B. B. Prog. Neurobiol. 1999,
59, 355.
5. Sebastiao, A. M.; Ribeiro, J. A. Prog. Neurobiol. 1996, 48, 167.
6. Fuxe, K.; Marcellino, D.; Genedani, S.; Agnati, L. Mov. Disord. 2007, 22, 1990.
7. Jenner, P. Neurology 2003, 61, S32.
8. Hauser, R. A.; Schwarzschild, M. A. Drugs Aging 2005, 22, 471.
9. Aoyama, S.; Kase, H.; Borrelli, E. J. Neurosci. 2000, 20, 5848.
10. Chen, J. F.; Sonsalla, P. K.; Pedata, F.; Melani, A.; Domenici, M. R.; Popoli, P.;
Geiger, J.; Lopes, L. V.; Mendonça, A. Prog. Neurobiol. 2007, 83, 310.